Many of the most valuable pharmaceuticals on the market or undergoing clinical trials are glycoproteins. Because these molecules are considerably more complex than traditional pharmaceuticals, they must be manufactured in mammalian cell lines. This imposes very significant costs on the manufacturer and introduces a large degree of heterogeneity into product formulations. To a large extent, both problems can be addressed by optimizing mammalian cell lines for the production of therapeutic proteins using genetic engineering techniques. Precision BioSciences develops "custom" homing endonucleases for use as genome engineering reagents. This proposal describes a proof-of-principle project to use Precision's genetic engineering technology to optimize Chinese hamster ovary (CHO) cells for the production of protein pharmaceuticals. Specifically, we intend to address the problem of low and inconsistent product yield by developing reagents and cells with which the gene encoding a bioproduct can be integrated into a specific location in the CHO genome (the DHFR locus) that is known to be resistant to gene silencing. In addition, we aim to demonstrate that engineered homing endonucleases can be used to manipulate the CHO glycosyltransferase repertoire with the long-term goal of generating a bioproduction platform comprising ~20 different cell lines with precisely engineered glycosylation properties. Because N-linked glycans play a major role determining the physiological effects of therapeutic glycoproteins, particularly antibodies, a set of cell lines which are capable of generating highly controlled glycoforms would have a major impact on the efficacy of protein therapeutics. PUBLIC HEALTH RELEVANCE: Many of the most beneficial pharmaceuticals in existence are proteins which, because of their complexity, are expensive to produce and difficult to perfect. We propose to use genetic engineering technology to produce mammalian cell lines that are optimized to be "factories" for the inexpensive production of higher quality protein pharmaceuticals.